Codon-optimized CFTR MRNA

Abstract

The present invention provides, among other things, improved methods and pharmaceutical compositions for treating cystic fibrosis based on codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein.

We claim: 
     
       1. A pharmaceutical composition for treating cystic fibrosis, comprising a codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1. 
     
     
       2. The pharmaceutical composition of  claim 1 , wherein the codon optimized CFTR mRNA encoding the CFTR protein is encapsulated within a nanoparticle. 
     
     
       3. The pharmaceutical composition of  claim 2 , wherein the nanoparticle is a liposome. 
     
     
       4. The pharmaceutical composition of  claim 3 , wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. 
     
     
       5. The pharmaceutical composition of  claim 3 , wherein the liposome comprises no more than three distinct lipid components. 
     
     
       6. A method of producing codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, comprising in vitro synthesizing codon optimized CFTR mRNA using a SP6 RNA polymerase,
 wherein at least 80% of the synthesized codon optimized CFTR mRNA molecules are full-length and wherein at least 100 mg of codon optimized mRNA is synthesized at a single batch. 
 
     
     
       7. The method of  claim 6 , wherein the in vitro synthesis of codon optimized CFTR mRNA results in a secondary polynucleotide species that constitutes less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the total mRNA synthesized. 
     
     
       8. The method of  claim 6 , wherein at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the synthesized codon optimized CFTR mRNA molecules are full-length. 
     
     
       9. The method of  claim 6 , wherein at least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 150 g, 200 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of codon optimized CFTR mRNA is synthesized at a single batch. 
     
     
       10. The method of  claim 6 , wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1. 
     
     
       11. The method of  claim 6 , wherein the method further comprises a step of capping and/or tailing of the synthesized codon optimized CFTR mRNA. 
     
     
       12. A pharmaceutical composition for treating cystic fibrosis, comprising an mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the mRNA encoding the CFTR protein comprises a polynucleotide sequence at least 85% identical to any one of SEQ ID NO: 21-30, 33, 34, 38-40. 
     
     
       13. The pharmaceutical composition of  claim 12 , wherein the mRNA is encoded in a nanoparticle, and wherein the nanoparticle is a liposome. 
     
     
       14. The pharmaceutical composition of  claim 13 , wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.